Vascular targeting of nanocarriers: perplexing aspects of the seemingly straightforward paradigm

Abstract

Targeted nanomedicine holds promise to find clinical use in many medical areas. Endothelial cells that line the luminal surface of blood vessels represent a key target for treatment of inflammation, ischemia, thrombosis, stroke, and other neurological, cardiovascular, pulmonary, and oncological conditions. In other cases, the endothelium is a barrier for tissue penetration or a victim of adverse effects. Several endothelial surface markers including peptidases (e.g., ACE, APP, and APN) and adhesion molecules (e.g., ICAM-1 and PECAM) have been identified as key targets. Binding of nanocarriers to these molecules enables drug targeting and subsequent penetration into or across the endothelium, offering therapeutic effects that are unattainable by their nontargeted counterparts. We analyze diverse aspects of endothelial nanomedicine including (i) circulation and targeting of carriers with diverse geometries, (ii) multivalent interactions of carrier with endothelium, (iii) anchoring to multiple determinants, (iv) accessibility of binding sites and cellular response to their engagement, (v) role of cell phenotype and microenvironment in targeting, (vi) optimization of targeting by lowering carrier avidity, (vii) endocytosis of multivalent carriers via molecules not implicated in internalization of their ligands, and (viii) modulation of cellular uptake and trafficking by selection of specific epitopes on the target determinant, carrier geometry, and hydrodynamic factors. Refinement of these aspects and improving our understanding of vascular biology and pathology is likely to enable the clinical translation of vascular endothelial targeting of nanocarriers.

Figure 1

Drug delivery by nanocarriers: optimization of drug pharmacokinetics and biodistribution (PK and BD). (A) PK and BD of drugs (blue dots) vs drugs encapsulated in nanocarriers. Free drugs are eliminated from blood by clearing organs, including the reticuloendothelial system (RES, including liver, spleen, and lymphatic nodes) and excretory organs such as kidneys, lungs, and the bile tract (including hepatic uptake and renal filtration), and diffusion in nontarget tissues including the brain (CNS), where drugs may cause adverse effects. Long-circulating nanocarriers avoiding clearance alter PK (large arrows depict sustained drug circulation) and reduce diffusion in nontarget tissues, thereby improving BD and inhibiting adverse effects. Relative dimensions in this and other cartoons and schemas are not to scale. (B and C) Panels represent, respectively, short-term and long-term model graphs of blood level of free vs nanocarrier-bound drugs (NC). After a single injection in acute and subacute conditions, long-circulating NCs enhance area under the curve, thus reducing the effective dose (B). Hypothetically, using NCs with extended lifetime in circulation will help to maintain a stable therapeutic dose without the need for repeated injections (C).

Figure 2

Passive uptake vs active targeting: differences in mechanism and potential biomedical utility. (A) Untargeted nanocarriers with size ranging from a few to a few hundred nanometers do not normally accumulate in healthy tissue with blood vessels lined by the continuous endothelial layer lacking large fenestrae typical of the reticuloendothelial system (RES). (B) Enhanced Permeation and Retention (EPR) effect. In this scenario, carriers accumulate in tissues with abnormally permeable vessels, such as in tumors fed by leaky vasculature as well as in sites of inflammation and angiogenesis (for example, wound healing). In tumors, deficient lymphatic drainage also favors the EPR effect, while high interstitial pressure opposes it (not shown). (C) Large particles, such as rigid spheres with diameter 10–50 μm (bigger than that of capillaries and precapillary arterioles), are mechanically retained downstream of the site of arterial injection in the microvasculature of an organ or tissue fed by this conduit artery. (D) Active targeting of nanocarriers coated by affinity ligands of specific determinants favors binding to endothelial cells exposing these determinants. In contrast with passive uptake (B and C), this mode guides subcellular delivery: binding to noninternalizable molecules vs those involved in cellular uptake and trafficking, respectively, favors retention on cell surface vs intracellular or transcellular delivery.

Figure 3

The vascular endothelium: a victim, barrier, and target of drug delivery. (A) In drug delivery strategies requiring cargoes to be released or act in the bloodstream, such as long-circulating reactors or slow release systems, respectively, carrier interaction with endothelium must be avoided. Otherwise, carriers adherence to endothelium may lead to vascular occlusion, endothelial damage, or pathological activation. (B) In strategies delivering drugs to the extravascular sites, endothelium is a barrier. Carriers may cross it by concentration gradient via large opening between endothelial cells in the RES (e.g., fenestrae) and intercellular openings in abnormally leaky vessels in tumors and sites of inflammation and angiogenesis (EPR). Vesicular transendothelial pathways include fluid phase transport via pinocytosis and transendothelial vacuolar–vesicular organelle (VVO) that opens from the caveolae. (C) In strategies targeting drug carriers to the endothelial surface determinants, ligand-mediated anchoring may result in surface retention or internalization. Depending on the nature of anchoring molecule, choice of epitopes, and carrier design, internalization may lead to recycling to the vascular lumen, delivery to the intracellular compartments, or transfer across the endothelium.

Figure 4

Balancing the stealth and avidity features of carriers: effect on targeting and PK. (A) Surface modification by hydrophilic polymeric chains (e.g., PEG) provides a carrier with stealth features but affects targeting via masking affinity ligands. Conjugation of ligand molecules to the end groups of polymeric chains instead of the carrier surface helps to avoid this negative interference and provides additional steric freedom for ligand–target interaction, thereby boosting binding. (B) Ligand molecules conjugated to PEG diminish its stealth effect via nonspecific interactions (e.g., mediated by altered charge or hydrophilic features) and ligand-mediated interactions (e.g., via Fc-fragment of antibodies conjugated to PEG). Ensuing acceleration of blood clearance may affect drug delivery, if a carrier’s circulation time is insufficient for targeting.

Figure 5

Carrier geometry modulates cellular uptake. Rate of intracellular uptake of elongated particles by phagocytic and other cells is markedly modulated by the axis of the particle binding to cell surface: it is faster than uptake of spherical particles of comparative maximal dimension in cases of binding via pointed vs flat aspects, respectively.

Figure 6

Endothelial determinants in normal and pathological vasculature: accessibility, regulation, and localization in specific domains of the plasmalemma. (Upper panel) Normal endothelium exposes determinants that are differently accessible to circulating carriers. They include molecules clustered in apical plasmalemma, such as ACE (A); located in the caveoli, such as APP and PV1 (B); molecules, such as ICAM-1, expressed as monomers and oligomers at basal level throughout the plasmalemma (C); molecules preferentially localized in membrane domains (e.g., lipid rafts, LR) (D); molecules partially masked by glycocalyx (E); and molecules concentrated in cellular junctions, such as PECAM and VE-cadherin (F). (Lower panel) Under pathological conditions, determinants may be masked by adherent white blood cells (WBC) and/or shed from the cells (A) (both mechanisms would inhibit targeting), whereas inducible adhesion molecules may exteriorize from intracellular stores (B), such as P-selectin from Weibel-Palade bodies (WPB), or be synthesized de novo, such as E-selectin, ICAM-1, and VCAM-1 (C). Pathological mediators also induce rearrangement of natural clusters (D); shedding of glycocalyx, thereby exposing normally masked determinants (E); and cause endothelial contraction, thereby increasing pericellular permeability and accessibility of target determinants in the junctions (F), such as VE-cadherin and PECAM.

Figure 7

Carrier shape and plasticity modulate ligand-mediated anchoring on endothelium. (A) Spherical carriers bind to the endothelium in a fashion reminiscent of the phases of leukocyte adhesion (rolling, initial tethering, and firm adhesion), whereas elongated and discoid carriers “zip up” on the target molecules. (B) Endothelial binding of flexible carriers is advantageous over that of rigid carriers, because shape change and lateral diffusion of ligands afford better congruency between molecules of a ligand/receptor pair and a higher number of productive anchoring engagements.

Figure 8

Maximal ligand surface density does not necessarily provide optimal carrier targeting. (A) Suboptimally low ligand density negatively impacts a carrier’s ability to engage in multivalent binding, hence suboptimal targeting as depicted in the model graph. (B) Surface density of ligand copies at which they engage multiple binding sites and achieve firm binding is sufficient and optimal for targeting. (C) Excessively high surface density of ligand molecules may lead to “a ligand overcrowding” phenomenon, i.e., inhibition of multivalent engagement due to steric limitations and competition of adjacent ligand molecules for the binding sites.

Figure 9

Controlled reduction of avidity (ligand surface density) enhances selectivity of carrier targeting to pathological endothelium. ICAM-1 is exposed on quiescent and pathological endothelium at modest basal and elevated levels, respectively. Carrier avidity to ICAM-1 is controlled by antibody surface density. High-avidity particles (approximately 150 nm diameter) carrying 200 molecules of anti-ICAM effectively bind to both quiescent and inflamed endothelial cells and show high pulmonary uptake after intravascular injection in both naïve and endotoxin-challenged mice (upper cartoons). In contrast, low avidity particles carrying 50 anti-ICAM molecules show negligible binding to quiescent cells, whereas elevation of surface density of ICAM-1 typical of pathological endothelium allows their multivalent anchoring and binding to cytokine activated endothelium (lower cartoons). This phenomenon helps to explain the several-fold higher selectivity of targeting to pathological vs normal endothelium demonstrated by low-avidity vs high-avidity carriers in mouse model of lung inflammation (middle panels). As result, low-avidity anti-ICAM coated carriers provided PET images of the pulmonary inflammation in endotoxin-challenged mice that were more discernible from naïve animals than images provided by high-avidity carriers (far-right panels). PET imaging at 1 h after IV injection of ICAM-targeted [¹²⁴I] carriers carrying either 200 or 50 antibody molecules per particle (upper vs lower images, respectively) in naïve vs endotoxin-treated mice (left vs right). White dashed-line corresponds to lung space. (Adapted with permission from ref (156). Copyright 2013 American Chemical Society.)

Figure 10

Carrier targeting using multiple ligands. (A) Carriers coated with ligands binding to endothelial determinants that are expressed in the area of interest selectively but scarcely (green) may have insufficient avidity for anchoring in this area. (B) Carriers coated with ligands binding to abundant endothelial determinants (red) anchor indiscriminately throughout the vasculature. (C) Carriers coated with a combination of two ligands may exert elevated basal avidity to endothelium, insufficient to provide a firm adhesion on its own but enabling binding in the case of simultaneous engagement of the scarce site-specific determinant.

Figure 11

Optimization of transcellular transport via controlled reduction of carrier avidity. (Upper panel) Carriers with high avidity to a receptor involved in endocytosis and transcellular transport more effectively bind to and enter endothelium than carriers with low avidity (lower panel). However, high-avidity carriers less effectively dissociate from the receptor after the internalization, which may impede transfer to the tissue. In this simplified cartoon, the high and low avidity of carriers is depicted as proportional surface density of a ligand, whereas avidity may also be regulated by different affinity of the ligands coated at similar density.

Figure 12

CAM-mediated endocytosis. Clustering cellular CAMs by multivalent anti-CAM conjugates activates a specific set of signaling kinases and the sodium/proton exchanger, NHE1, which leads to formation of actin stress fibers needed for the uptake of endocytic vesicles. Internalized carriers traffic to endosomes, where engaged CAM molecules dissociate from the carriers and recycle to the plasma membrane. Several hours after internalization, carriers arrive at lysosomes where proteolysis-sensitive carriers and cargoes are degraded. Nocodazole (which disrupts the cell microtubule network), chloroquine (which inhibits lysosomal acidification), and monensin (which enhances Na^+/H⁺ exchange in endosomes and induces recycling of anti-CAM nanoconjugates to the plasma membrane) modulate intracellular traffic and effects of CAM-targeted DDS. (Reproduced with permission from B. Ding et al., Advanced Drug Delivery Systems That Target the Vascular Endothelium, Mol. Interv.2006, 6, 98–112.)

Figure 13

Flow-mediated modulation of CAM endocytosis. Endothelium in vivo is constantly or intermittently exposed to blood flow and chronic and acute exposure to flow differentially regulates endocytosis of CAM-targeted carriers. (A) Endothelial adaptation to chronic flow, manifested by cellular alignment with flow direction and formation of actin stress fibers, inhibits anti-CAM/carrier endocytosis, likely via impaired recruitment of actin in the stress fibers needed for endocytosis. (B) In contrast, acute exposure of endothelium to flow (imitating reperfusion of exertion perfusion) stimulates endocytosis of carriers, likely through mechanisms involving signaling via cholesterol-rich domains of plasmalemma such as caveoli (cav).

Figure 14

Enhanced blood level of low-avidity carriers as an additional factor facilitating cellular transport. PEGylated nanocarriers coated with a ligand at high density are more rapidly eliminated from blood by the clearing organs (e.g., liver) than carriers coated by a ligand at lower density (and thus retaining more preserved stealth features). This inequity in the PK may help to explain more effective cellular uptake and transport of low-avidity carriers due to elevated concentration in blood favoring binding to the target determinants.